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Biochemical reaction of Gram

negative bacteria

Sharq Elneil College

School of Medical Laboratory Sciences

Department of Microbiology

Medical Bacteriology course

U.Mahadi Hassan Mahmoud Bsc, Msc, MIBMS Microbiology

INDOLE TEST

Principle:

Certain bacteria break down Amino acid called Tryptophan to Give Indole. Indole react with Covac’s reagent to give Red ring.

Method:

Sub-culture on media contain Tryptophan (e.g.: Peptone water, Incubate at 37ºC for 24 hrs.

Add few drops of Covac’s Reagent (4-paradimethyle amino benzaldehyde in amyl alcohol and HCL).

Examine for development of Red Ring.

+ve E. coli, -ve Klebsiella

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Methyl Red (MR)

Principle:

Certain bacteria ferment Glucose to give high

amount of Lactic acid which react with Methyl

Red to give Red colour.

Method:

Sub-culture on Glucose Phosphate peptone

water, incubate at 37ºC for 24 hrs.

Add few drops from MR and see development

of Red colour.

+ve E. coli, -ve Klebsiella.

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Vogous Proskaur (V.P)

Principle:

Some organism Breakdown Glucose to give Acetyl Methyl carbinol which oxidized by air to give Diacetyl methyl carbinol react with -naphthol in Alkaline pH (KOH) to give red colour.

Method:

Subculture in Glucose Phosphate peptone water incubate at 37ºC for 24 hrs.

Add 1 ml of 40% KOH and 3 ml - naphthol and examined for red colour with 2-5 min.

Listeria Monocytogenes V.P +ve and MR +ve.

Citrate Utilization test

Media content:

Koser’s citrate: (Broth media contain ammonium citrate and bromothymol blue)

Simon Citrate (Koser’s citrate + agar)

Principle:

Some organism Utilize citrate as a source of carbon allow the ammonium ion free (Alkaline pH) this give blue colour. Method:

Subculture on media contain ammonium citrate and incubate then read after 24-48 hrs. may take 96 hrs.

Proteus Citrate +ve, E. coli Citrate –ve.

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Urease test

Principle:

Some organism secrete Urease which breakdown urea into ammonia (Alkaline) which change the colour of phenol red into red colour.

Method:

Subculture on christensen’s urea broth or urea agar slop, incubate for 4 hrs and see the development of red colour.

Proteus is rapidly urease +ve, E. coli is Urease -ve

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Oxidase test

Principle: Certain organism produce oxidase enzyme that

oxidize oxidase reagents to give purple colour.

Methods:

Filter paper method: Test requirements:

Freshly prepared 1% Oxidase reagent (tetramethyl-p-phenylene diamine).

Filter paper.

Wood stick or glass rods.

Take a colonies and put it in filter paper, add drop of oxidase reagents and examined for purple colour.

Oxidase +ve like Neisseria and Pseudomonas.

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H2S production

Principle:

H2S react with Ferric ammonium citrate to

give black colour.

Method:

Subculture the organism in any media (solid

or broth) and put on the cover filter paper

impregnated on Ferric ammonium citrate,

Examined for black colour after 24 hrs

incubation.

Proteus and Salmonella H2S +ve.

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Motility test

Kligler Iron agar

Principle: This test used to detect the ability of the organism to

ferment Glucose, or Glucose and Lactose with the production of Acid only or with Acid Which react with ferric chloride to give blacking.

Glucose fermentation give lactic acid which oxidized by air and return back to glucose on the top of the medium.

Media content: Nutrition, Ferric chloride, Phenol red, Glucose and

Lactose.

Method: Subculture using straight loop till reach the bottom

(Butt) and get out making Zigzag on the surface, incubate and read.

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Sugar fermentation

Principle: The ability of the organism to ferment sugar depends

on production of two enzymes:

Permiase----Allow the penetration of sugar.

β Galactosidase----Ferment the sugar.

Media content: Nutrient broth, Sugar (Glucose, Maltose,

sucrose,….), Durham tube, Andrad’s indicator (Acid Fuchsin neutralized by NaOH).

Method: Subculture, incubate, read.

Acid---- Yellow colour.

Alkaline---- Red colour.

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ONPG

Principle:

Some organism produce β Galactosidase only (Late lactose fermenter). This enzymes detected by ONPG (O-NitroPhenyl-β-D-Galactoside) test in which β Galactosidase break down β Galactoside allow the O-NitroPhenyl free which have yellow colour.

Method:

Subculture the organism on broth media contain O-Nitrophenyl-β-D-Galactoside, incubate and read for formation of Yellow colour.

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Oxidation Fermentation

Principle: The test depends on fermentation of carbohydrate on

anaerobic condition of oxidation of it in aerobic condition.

Media content: 2 media each contain nutrition, sugar and bromothymol

blue. One of them closed from air by paraffin oil.

Results: Oxidative ferment sugar on open tube (Yellow colour).

Fermentative but anaerobically give yellow on closed tube.

Facultative anaerobic ferment CHO on both tube (Yellow).

Non oxidative- Non fermentative– give Blue colour on both tube.

Phenyl Pyruvic Acid (PPA)

Principle:

Some organism deaminate Phenyl alanine to Phenyl Pyruvic acid which react with Ferric chloride to give green colour.

Method:

Subculture the organism on media contain Phenyl alanine and incubate at 37C for 24 hrs, add 10% ferric chloride on the surface and examined for green colour.